The present invention is directed to compositions and methods for producing avermectins, and is primarily in the field of animal health. More particularly, the present invention relates to the identification and characterization of two novel genes, herein referred to as the aveR1 and aveR2 genes, that are involved in regulating avermectin polyketide synthase (PKS) expression and avermectin biosynthesis in Streptomyces avermitilis. The present invention is based on the discovery that inactivation of these genes results in an increase in the amount of avermectin produced by S. avermitilis. 
Streptomyces species produce a wide variety of secondary metabolites, including the avermectins, which comprise a series of eight related sixteen-membered macrocyclic lactones with potent anthelmintic and insecticidal activity. The eight distinct but closely related compounds are referred to as A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. The xe2x80x9caxe2x80x9d series of compounds refers to the natural avermectin wherein the substituent at the C25 position is (S)-sec-butyl, and the xe2x80x9cbxe2x80x9d series refers to those wherein the substituent at the C25 position is isopropyl. The designations xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d refer to avermectins wherein the substituent at C5 is methoxy and hydroxy, respectively. The numeral xe2x80x9c1xe2x80x9d refers to avermectins wherein a double bond is present at the C22, 23 position, and the numeral xe2x80x9c2xe2x80x9d refers to avermectins having a hydrogen at the C22 position and a hydroxy at the C23 position. Among the related avermectins, the B1 type of avermectin is recognized as having the most effective antiparasitic and pesticidal activity, and is therefore the most commercially desirable avermectin.
The avermectins and their production by aerobic fermentation of strains of S. avermitilis are described, among other places, in U.S. Pat. No. 4,310,519 and 4,429,042.
The avermectin (ave) genes, like many genes involved in the production of secondary metabolites and other Streptomyces antibiotics, are found clustered together on the bacterial chromosome. The ave gene cluster for avermectin biosynthesis spans a 95 kb genomic fragment of DNA which includes DNA encoding the avermectin polyketide synthase (PKS) (MacNeil et al., 1992, Gene 115:119-125).
The regulation of antibiotic biosynthesis in Streptomyces is perhaps best characterized in the species Streptomyces coelicolor. Four antibiotics produced by S. coelicolor include actinorhodin (Act), undecylprodigiosin (Red), calcium-dependent antibiotic (CDA), and methylenomycin (Mmy). Each of these antibiotics is encoded by a different cluster of genetically distinct genes. Genes have been identified that are linked to either the Act gene cluster or the Red gene cluster that encode products which specifically regulate the expression of the Act biosynthetic gene cluster or the Red biosynthetic gene cluster, respectively. A number of loci containing genes that globally regulate more than one of the antibiotic biosynthetic gene clusters have also been identified. For example, mutations in two independent loci, absA and absB, have been shown to block the synthesis of all four antibiotics in S. coelicolor (Brian et al.; 1996, J. Bact. 178:3221-3231). The absA locus has been cloned and characterized, and its gene products have been shown to be involved in a signal transduction pathway which normally acts as a global negative regulator of antibiotic synthesis in S. coelicolor (Brian et a., 1996, above).
U.S. Pat. No. 5,876,987 to Champness et al. relates to hyperproduction of antibiotic in Streptomyces spp. as a result of interruption of the absA locus.
U.S. Pat. No. 5,707,839 to Denoya, and U.S. Pat. No. 5,728,561 to Denoya et al. relate to DNA sequences encoding branched-chain alpha-ketoacid dehydrogenase complexes of Streptomyces and methods for enhancing the production of novel avermectins.
Understanding the mechanism by which Type I polyketide synthase expression is regulated in S. avermitilis will permit genetic manipulation of the ave genes to increase the production of avermectins.
The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an aveR1 gene product from S. avermitilis. In a preferred embodiment, the aveR1 gene product comprises the amino acid sequence of SEQ ID NO:2. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317. In a further non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:1.
The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO:2.
The present invention further provides an isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned aveR1-related polynucleotide molecules of the present invention. In a preferred embodiment, the substantial portion of the aveR1-related polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of a S. avermitilis aveR1 gene product or aveR1-related homologous polypeptide of the present invention. In a specific though non-limiting embodiment, the present invention provides a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment consisting of a sub-sequence of the amino acid sequence of SEQ ID NO:2.
The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR1 ORF of S. avermitilis in situ. Such flanking sequences can be selected from the nucleotide sequence of SEQ ID NO:1 from about nt 1 to about nt 1111, and from about nt 2318 to about nt 5045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous to nucleotide sequences that naturally flank the aveR1 ORF of S. avermitilis in situ. Each flanking sequence, or homolog thereof, in the isolated polynucleotide molecule of the present invention is preferably at least about 200 nt in length. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences that naturally flank the aveR1 ORF of S. avermitilis in situ, or that are homologous to such nucleotide sequences, and further comprising one of the aforementioned aveR1-related nucleotide sequences of the present invention such as, e.g., the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317 or substantial portion thereof.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an aveR2 gene product from S. avermitilis. In a preferred embodiment, the aveR2 gene product comprises the amino acid sequence of SEQ ID NO:4. In a non-limiting embodiment, the isolated polynucleotide molecule of the present inventon comprises the nucleotide sequence of the aveR2 ORF of S. avermitilis as shown in SEQ ID NO:3 (note: SEQ ID NO:3 is identical to SEQ ID NO:1) from about nt 2314 to about nt 3021. In a further non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:3.
The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence of the aveR2 ORF of S. avermitilis as shown in SEQ ID NO:3 from about nt 2314 to about nt 3021.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO:4.
The present invention further provides an isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned aveR2-related polynucleotide molecules of the present invention. In a preferred embodiment, the substantial portion of the aveR2-related polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of a S. avermitilis aveR2 gene product or aveR2-related homologous polypeptide of the present invention. In a specific though non-limiting embodiment, the present invention provides a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment consisting of a sub-sequence of the amino acid sequence of SEQ ID NO:4.
The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR2 ORF of S. avermitilis in situ. Such flanking sequences can be selected from the nucleotide sequence of SEQ ID NO:3 from about nt 1 to about nt 2313, and from about nt 3022 to about nt 5045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous to nucleotide sequences that naturally flank the aveR2 ORF of S. avermitilis in situ. Each flanking sequence in the isolated polynucleotide molecule of the present invention is preferably at least about 200 nt length. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences that naturally flank the aveR2 ORF of S. avermitilis in situ, or that are homologous to such nucleotide sequences, and further comprising one of the aforementioned aveR2-related nucleotide sequences of the present invention such as, e.g., the nucleotide sequence of the aveR2 ORF as shown in SEQ ID NO:3 from about nt 2314 to about nt 3021 or a substantial portion thereof.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding both the aveR1 and aveR2 gene products from S. avermitilis. In a preferred embodiment, the aveR1 and aveR2 gene products comprise the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4, respectively. In a non-limiting embodiment, the isolated polynucleotide molecule comprises the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317 and the aveR2 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 2314 to about nt 021. In a further non-limiting embodiment, the isolated polynucleotide molecule comprises he nucleotide sequence of SEQ ID NO:1 from about nt 1112 to about nt 3021. In a further on-limiting embodiment, the isolated polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO:1.
The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence of both the aveR1 and aveR2 ORFs of S. avermitilis. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317, and the aveR2 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 2314 to about nt 3021.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a first polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO:2 and a second polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO:4.
The present invention further provides an isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned polynucleotide molecules which comprise a nucleotide sequence encoding both the aveR1 and aveR2 gene products from S. avermitilis or any of the aforementioned polynucleotide molecules that are homologous thereto. In a specific though non-limiting embodiment, the substantial portion of the polynucleotide molecule consists of the nucleotide sequence of the aveR1 ORF as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317. In another specific though non-limiting embodiment, the substantial portion of the polynucleotide molecule consists of the nucleotide sequence of the aveR2 ORF as shown in SEQ ID NO:3 from about nt 2314 to about nt 3021.
The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR1 and aveR2 ORFs of S. avermitilis in situ. Such flanking sequences can be selected from the nucleotide sequence of SEQ ID NO:1 from about nt 1 to about nt 1111, and from about nt 3022 to about nt 5045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous to nucleotide sequences that naturally flank the aveR1 and aveR2 ORFs of S. avermitilis in situ. Each flanking sequence, or homolog thereof, in the isolated polynucleotide molecule of the present invention is preferably at least about 200 nt in length. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences that naturally flank the aveR1 and aveR2 ORFs of S. avermitilis in situ, or that are homologoous to such nucleotide sequences, and further comprising one of the aforementioned nucleotide sequences of the present invention that encode either or both of the aveR1 and aveR2 gene products from S. avermitilis, such as, e.g., the nucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317, and the nucleotide sequence of the aveR2 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 2314 to about nt 3021, and substantial portions thereof.
The present invention further provides oligonucleotide molecules that are useful as primers to amplify any of the aforementioned polynucleotide molecules of the present invention or portions thereof, or that can be used to encode or act as anti-sense molecules useful in regulating ave gene expression and avermectin production.
The present invention further provides compositions and methods for cloning and expressing any of the polynucleotide molecules or oligonucleotide molecules of the present invention, including cloning vectors, expression vectors, transformed host cells comprising any of said vectors, and novel strains or cell lines derived therefrom. In a non-limiting embodiment, the present invention provides a recombinant expression vector comprising a polynucleotide molecule of the present invention in operative association with one or more regulatory elements necessary for expression of the polynucleotide molecule. In a specific though non-limiting embodiment, the present invention provides plasmid pSE201 (ATCC 203182), which comprises the complete ORFs of both the aveR1 and aveR2 genes of S. avermitilis. Other plasmids are described below.
The present invention further provides a substantially purified or isolated polypeptide encoded by a polynucleotide molecule of the present invention. In a specific though non-limiting embodiment, the polypeptide is an aveR1 gene product comprising the amino acid sequence of SEQ ID NO:2. In another specific though non-limiting embodiment, the polypeptide is an aveR2 gene product comprising the amino acid sequence of SEQ ID NO:4. The present invention further provides substantially purified or isolated polypeptides that are homologous to either the aveR1 or aveR2 gene products of the present invention. The present invention further provides substantially purified or isolated peptide fragments of the aveR1 or aveR2 gene products or homologous polypeptides of the present invention.
The present invention further provides a method of preparing a substantially purified or isolated aveR1 gene product, aveR2 gene product, homologous polypeptide, or peptide fragment of the present invention, comprising culturing a host cell transformed or transfected with a recombinant expression vector of the present invention under conditions conducive to the expression of the particular encoded gene product, polypeptide, or peptide fragment, and recovering the expressed gene product, polypeptide, or peptide fragment from the cell culture.
The present invention further provides compositions and methods for genetically modifying the cells of a species or strain of Streptomyces, including genetic constructs such as, e.g., gene replacement vectors. As provided by the present invention, the cells of a species or strain of Streptomyces are genetically modified to produce an amount of avermectins which is detectably different from the amount of avermectins produced by cells of the same species or strain that have not been so modified. In a preferred embodiment, the cells of a species or strain of Streptomyces are genetically modified to produce a detectably increased amount of avermectins compared to the amount of avermectins produced by cells of the same species or strain that have not been so modified. In a further preferred embodiment, the species of Streptomyces is S. avermitilis. According to the present invention, such genetic modification preferably comprises mutating either an aveR1 homolog gene, or an aveR2 homolog gene, or both the aveR1 and aveR2 homolog genes, where such mutation results in a detectable increase in the amount of avermectins produced by cells of a strain of Streptomyces carrying the mutation compared to cells of the same strain that do not carry the gene mutation. Mutation of either the aveR1 homolog gene or the aveR2 homolog gene, or both aveR1 and aveR2 homolog genes, can be carried out using standard mutagenic techniques, including exposure to a chemical mutagen or radiation, or by using a genetic construct provided by the present invention, such as, e.g., a gene replacement vector, to mutate the aveR1 homolog gene or aveR2 homolog gene, or both aveR1 and aveR2 homolog genes, by, e.g., adding, deleting or substituting nucleotides, or by introducing a frame-shift, or by inserting a different or heterologous nucleotide sequence into the aveR1 homolog gene or aveR2 homolog gene, or by deleting a portion or all of either the aveR1 homolog gene or the aveR2 homolog gene, or both the aveR1 and aveR2 homolog genes, or by replacing a portion or all of either the aveR1 homolog gene or the aveR2 homolog gene, or both the aveR1 and aveR2 homolog genes, with a different or heterologous nucleotide sequence, or by a combination of such mutations.
The present invention further provides a method for identifying a mutation of an aveR1 homolog gene or aveR2 homolog gene, or of both aveR1 and aveR2 homolog genes, in a species or strain of Streptomyces, which mutation is capable of detectably increasing the amount of avermectins produced by cells of the species or strain of Streptomyces carrying the gene mutation compared to cells of the same species or strain of Streptomyces that do not carry the gene mutation, comprising: (a) measuring the amount of avermectins produced by cells of the particular species or strain of Streptomyces;. (b) introducing a mutation into the aveR1 homolog gene or aveR2 homolog gene, or into both the aveR1 and aveR2 homolog genes, of cells of the species or strain; and (c) comparing the amount of avermectins produced by the cells carrying the gene mutation as produced in step (b) to the amount of avermectins produced by the cells of step (a) that do not carry the gene mutation; such that if the amount of avermectins produced by the cells carrying the gene mutation as produced in step (b) is detectably higher than the amount of avermectins produced by the cells of step (a) that do not carry the gene mutation, then a mutation of the aveR1 or aveR2 homolog gene, or of both the aveR1 and aveR2 homolog genes, capable of detectably increasing the amount of avermectins has been identified. In a preferred embodiment, the species of Streptomyces is S. avermitilis. 
The present invention further provides a method of preparing genetically modified cells from a particular species or strain of Streptomyces, which modified cells produce a detectably increased amount of avermectins compared to unmodified cells of the species or strain, comprising mutating the aveR1 homolog gene or the aveR2 homolog gene, or both the aveR1 and aveR2 homolog genes, in cells of the species or strain of Streptomyces, and selecting those cells which produce a detectably increased amount of avermectins as a result of the mutation compared to cells of the same species or strain of Streptomyces that do not carry the gene mutation. In a preferred embodiment, the species of Streptomyces is S. avermitilis. In a specific though non-limiting embodiment described below in Section 6.9.1, both the aveR1 and aveR2 genes of S. avermitilis were mutated by replacing a portion of the ORF of each gene with a heterologous gene, resulting in S. avermitilis cells that produce a detectably increased amount of avermectins compared to cells of the same strain of S. avermitilis in which the aveR1 and aveR2 genes have not been so mutated. In another specific though non-limiting embodiment described below in Section 6.9.2, the aveR2 gene of S. avermitilis was mutated by inserting a heterologous gene into the aveR2 ORF, resulting in S. avermitilis cells that produce a detectably increased amount of avermectins compared to cells of the same strain of S. avermitilis in which the aveR2 gene has not been so mutated.
The present invention further provides novel strains of Streptomyces, the cells of which produce a detectably increased amount of avermectins as a result of one or more mutations to the aveR1 homolog gene or aveR2 homolog gene, or to both the aveR1 and aveR2 homolog genes, compared to cells of the same strain of Streptomyces that do not carry the gene mutation. In a preferred embodiment, the strain of Streptomyces is from the species S. avermitilis. The novel strains of the present invention are useful in the large-scale production of avermectins, such as the commercially desirable doramectin.
The present invention further provides a process for increasing the amount of avermectins produced by cultures of Streptomyces, comprising culturing cells of a particular species or strain of Streptomyces, which cells comprise a mutation in the aveR1 homolog gene or aveR2 homolog gene, or in both the aveR1 and aveR2 homolog genes, and which gene mutation serves to detectably increase the amount of avermectins produced by cells of the species or strain of Streptomyces carrying the gene mutation compared to cells of the same species or strain that do not carry the gene mutation, in culture media under conditions which permit or induce the production of avermectins therefrom, and recovering the avermectins from the culture. In a preferred embodiment, the species of Streptomyces is S. avermitilis. This process is useful to increase the production efficiency of avermectins.
The present invention further provides antibodies directed against an aveR1 gene product, aveR2 gene product, homologous polypeptide, or peptide fragment of the present invention.